Most anti-neoplastic drugs have a narrow therapeutic window. Bone marrow suppression is frequently the major toxicity. We propose a combined basic and translational research program to establish new, more efficacious mutant enzymes for the protection of bone marrow in patients undergoing cancer chemotherapy. Our objective is to express the mutant enzymes in bone marrow stem cells to achieve greater protection against the toxicity of frequently administered chemotherapeutic agents than is afforded by the wild-type enzymes. Our approach is to utilize human enzymes involved in different aspects of DNA metabolism. Mutant O6-methylguanine - DNA methyltransferase (MGMT), acting at the level of DNA repair, will be used to increase resistance to alkylating agents. Mutant DNA polymerase eta (pol eta; pol eta) that can more efficiently bypass chemical modifications in DNA will be studied as a prototype polymerase for enhancing tolerance of unrepaired DNA lesions. Mutant, drug-resistant thymidylate synthase (TS), acting at the level of precursor formation for DNA synthesis, will be used to protect against the widely utilized substrate analog inhibitor 5-fluorouracil (5-FU). Mitigation of myelosuppression has tremendous potential benefits, including (1) adherence to regimens that are now interrupted or abandoned due to marrow toxicity, (2) toleration of higher drug doses than are currently employed, and (3) utilization of drug combinations that are too marrow-toxic to be useful at present.